1. Field of the Invention
The present invention relates generally to a novel chymotrypsin that exhibits resistance to a plant serine proteinase inhibitor. More particularly, the present invention provides a chymotrypsin which is up-regulated in the gut of Helicoverpa armigera and Helicoverpa punctigera insect larvae when fed the serine proteinase inhibitors of Nicotiana alata. The novel chymotrypsin represents, therefore, a target for the identification of antagonists including inhibitors which are proposed to be useful in the control of Helicoverpa spp. populations that have become resistant to serine proteinase inhibitors produced in plants. The antagonists of the chymotrypsin may be topically applied to the plants or, when in proteinaceous form, may be produced by genetic means in plant cells. The antagonists may act at the level of gene expression or protein activity.
2. Description of the Prior Art
Bibliographic details of the publications referred to in this specification are also collected at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Female reproductive tissues and wounded leaves of the ornamental tobacco, Nicotiana alata amass high levels of serine proteinase inhibitors (PIs) for protection against pests and pathogens (Atkinson et al., The Plant Cell 5: 203-213, 1993). These 6 kDa PIs accumulate in the vacuole (Miller et al., Plant Cell 11: 1499-1508, 1999) and are derived in vivo from the post-translational modification of a 40.3 kDa precursor protein. The precursor of the PI protein (referred to as “NaPI” ) is composed of six repeated regions of high sequence identity (FIG. 1) each with a potential PI reactive site. Processing of the six-repeat precursor protein unexpectedly occurs at sites located within, rather than between the repeated regions. Complete removal of the linker sequence (Glu-Glu-Lys-Lys-Asn) [SEQ ID NO:1] contained within each repeated region, generates five contiguous 6 kDa inhibitors (C1 and T1-T4) and a novel two-chain chymotrypsin inhibitor (C2) formed by disulphide bond linkage of N-terminal and C-terminal peptide fragments (Heath et al., European Journal of Biochemistry 230(1): 25-257, 1995; Lee et al., Nature Structural Biology 6(6): 526-530, 1999). The structures of C1, T1-T4 and C2 have been solved using 1H-NMR techniques (Nielson et al., J. Mol. Biol. 242: 231-243, 1994; Nielson et al., Biochemistry 34: 14304-14311, 1995; Lee et al., 1999, supra).
Nicotiana alata also has a second gene related to NaPI that encodes a closely related precursor protein with four rather than six repeated domains (Miller et al., Plant Mol. Biol. 42: 329-333, 2000). This precursor is also processed in vivo resulting in the release of three contiguous 6 kDa inhibitors (C1, T4 and T5) and the two-chain inhibitor C2 (FIG. 1). Three of the inhibitors (C1, C2 and T4) are identical to those released from the six-domain precursor. Related multidomain precursors have been described for other solanaceous plants including N. tabacum (Balandin et al., Plant Mol. Biol. 27: 1197-1204, 1995), N. glutinosa (Choi et al., Biochim. et Biophys. Acta 1492: 211-215, 2000), L. esculentum (Taylor et al., Plant Mol. Biol. 23: 1005-1014, 1993) and Capsicum annum (Moura and Ryan, Plant Physiol. 126: 289-298, 2001; Antcheva et al., Protein Sci. 10: 2280-2290, 2001).
Several groups have reported on the affect of serine proteinase inhibitors on the activity of the digestive proteases of insects and have suggested that they are produced by plants for protection against the damaging affects of insect pests and microorganisms (Ryan, Annu. Rev. Phytopathol. 28: 425-449, 1990; Gatehouse et al., In: Plant Genetic Manipulation for Crop Protection, Biotech. in Agriculture No. 7, Eds. Gatehouse, Hilder & Boulter, International U.K., pp. 155-181, 1992). Insects that are specialist feeders on a particular host plant are generally resistant to the serine PIs produced by that plant, but are sensitive to PIs produced by non-hosts (Broadway and Villani, Entomol. Expo. Appl. 76: 303-312, 1995; Broadway, J. Insect. Physiol. 41: 107-116, 1995). There is interest, therefore, in transferring genes encoding serine PIs from non-hosts into crop plants to enhance insect resistance and to decrease reliance on chemical pesticides. Recently, however, several groups have reported on the ability of certain insects to change the relative proportions of proteolytic enzymes in their midgut following ingestion of high levels of PIs (Broadway, 1995, supra; Jongsma et al., Proc. Natl. Acad. Sci. USA 92(17): 8041-8045, 1995a). Broadway (1995, supra), for example, found that certain lepidopteran insects produce two broad classes of trypsin like proteases, one of which is insensitive to PIs from cabbage leaves. After ingestion of the cabbage PIs the insects increased production of the trypsin class not affected by the PIs and thus were able to grow and develop unhindered. Jongsma and coworkers (1995, supra) made a similar observation with Spodoptera exigua larvae fed on PIs from potato (PotII) and tobacco. The factors that regulate the secretion of these proteases under these conditions are not known.
These studies indicate that PIs specific for only one or two of the range of proteinases in an insect gut will be of limited use for long term plant protection. The gene encoding the N.alata PI has a potential advantage over other plant PIs for the enhancement of insect resistance in transgenic plants. Most plant serine PIs contain only one or two inhibitory domains, whereas the N. alata PI precursors have four or six (FIG. 1). Thus, there is potential to engineer the individual domains of the N. alata PI to provide inhibitory activity against several proteinases in the insect gut.
The midgut proteases of several Lepidoptera, Coleoptera and Orthoptera have been partially characterized. In most Lepidopteran species the endoproteinase activity is due primarily to serine proteinases (trypsin, chymotrypsin and/or elastase) and cysteine and metalloproteinases are not detectable (Christeller et al., Insect Biochem. Molecul. Biol. 22: 735-746, 1992; Terra and Ferreira, Comp. Biochem. Physiol. 109: 1-62, 1994; Xu and Qin, J. Econ. Entomol. 87: 334-338, 1994; Lee and Anstee, Insect. Biochem. Molec. Biol. 25: 63-71, 1995a; Johnston et al., Insect Biochem. 21: 389-397, 1991; Johnston et al., Insect Biochem. Molec. Biol. 25(3): 375-383, 1995). Exopeptidase and leucineaminopeptidase have also been identified (Christeller et al., 1992, supra; Lee and Anstee, Insect. Biochem. Molec. Biol. 25(1): 49-61, 1995b).
The mechanism of action of PIs on insects is only partially understood. Three responses have been described:                (i) Severe retardation of growth without a decrease in gut proteolytic activity. Broadway and Duffey (J. Insect Physiol. 32: 673-680, 1986a; Broadway and Duffey, J. Insect Physiol. 32: 827-833, 1986b) found that insects fed on PIs had remarkably reduced growth rates that were not associated with a decrease in the total proteolytic activity in the gut. Indeed the gut proteolytic activity often increased. They suggested that a feedback mechanism was operating that led to hyperproduction of proteases, that led in turn to a depletion of essential sulphur containing amino acids. This phenomenon has been recorded for other insects after chronic ingestion of PIs (Burgess et al., Entomol. Exp. App. 61: 123-130, 1991; De Leo et al., Plant Physiol. 118: 997-1004, 1998; Markwick et al., J. Economic Entomology 91 (6): 1265-76, 1998).        (ii) Severe retardation of growth with a decrease in gut proteolytic activity. Broadway (1995, supra) found that the lepidopteran species, Agrotis ipsilon (black cutworm) had reduced growth and delayed pupation after exposure to soybean trypsin inhibitor and did not respond by secreting PI-insensitive proteases. These insects had up to a 70% reduction in total gut proteolytic activity. Codling moth larvae (Lepidoptera:Tortricidae) fed on ‘elastase inhibitors’ were also retarded in growth and development that was associated with diminished elastase activity in the gut (Markwick et al., Journal of Economic Entomology 88(1): 33-39, 1995).        (iii) No effect on growth—change in the complement of gut proteinases. Some insects can compensate for the inhibition of one group of proteinases by inducing a new proteinase activity. The genomes of lepidopteran insects contain genes for a range of serine proteases and insects can modify the expression of specific isozymes to suit dietary components (Bown, et al., Insect Biochem. Molec. Biol. 27: 625-638, 1997; Broadway, J. Insect. Physiol. 43(9): 855-874, 1997). Changes in the complement of gut trypsins and chymotrypsins have been detected using Northern blot analysis on RNA from H. armigera (Bown, et al., 1997, supra; Gatehouse, et al., Insect Biochem. Molecul. Biol. 27: 929-944, 1997), H. zea and Agrotis ipsilon (Mazumdar-Leighton and Broadway, Insect Biochem. Mol. Biol. 31: 645-657, 2001a; Mazumdar-Leighton and Broadway, Insect Biochem. Mol. Biol. 31:633-644, 2001b). Corresponding changes at the protein level have also been observed using electrophoretic separation of isozymes for H. armigera (Harsulkar, et al., Plant Physiol. 121: 497-506, 1999; Patankar, et al., Insect Biochem. & Mol. Biol. 31: 453-464, 2001), Spodoptera frugiperda (Paulillo, et al., J. Econ. Entomol. 93:892-896, 2000), H. zea and Trichoplusia ni (Broadway, Arch. Insect Biochem. Physiol. 32(I): 39-53, 1996). Sometimes specific isozymes have been up-regulated, and occasionally proteases previously undetected have been observed.        
Recently, Mazumdar-Leighton and Broadway (2001a, supra) demonstrated that the production of PI-insensitive trypsins in H. zea is regulated at the transcriptional level and can be abolished using the transcriptional regulator actinomycin. Broadway and colleagues examined changes in gut trypsin and chymotrypsin activity after H. zea and Trichoplusia ni larvae were fed for 48 h on artificial diet containing 1% SBTI (Broadway, 1996, supra). Trypsin activity increased after SBTI consumption and protease banding patterns on zymograms indicated a change in the relative complement of proteases. The researchers showed in vitro that SBTI could inhibit 74% of the trypsin activity in gut extracts from control larvae, but only 3% of the gut trypsin activity in larvae that had consumed SBTI. They suggested the new protease bands (one new band for H. zea and 6 new bands for T. ni) on the zymograms may be SBTI-insensitive trypsins or SBTI-insensitive chymotrypsins and concluded that the production of these new proteases was enhanced by the ingestion of SBTI. Further studies using Northern blot analysis showed that consumption of SBTI resulted in transcriptional induction of mRNAs encoding trypsins and chymotrypsins by H. zea and Agrotis ipsilon (Mazumdar-Leighton and Broadway, 2001a, supra; Mazumdar-Leighton and Broadway, 2001b, supra), although it was not determined if these proteases were SBTI-insensitive.
The novel trypsin transcript induced in H. zea after ingestion of SBTI was designated HzT15 (Mazumdar-Leighton and Broadway, 2001b, supra). Recently, the first insect digestive enzyme insensitive to several proteinase inhibitors was purified from the gut of H. zea and corresponds to the protein encoded by HzT15 (Volpicella et al., Eur. J Biochem. 270: 10-19, 2003). The authors identified several differences in charge distribution across the surface of the structural model of this PI-insensitive trypsin relative to the PI inhibitable trypsins, but were unable to identify the structural changes that led to resistance.
Until recently, chymotrypsins were assumed to contribute relatively little to protein digestion in Lepidoptera and consequently most biochemical studies focused on characterization of the trypsins. This problem arose due to the initial use of synthetic substrates that worked well with mammalian chymotrypsins, but not at all or poorly with the Lepidopteran enzymes. Lepidopteran chymotrypsins prefer synthetic substrates with at least four amino acids to occupy the S1-S4 binding subsites on the enzyme, whereas mammalian trypsins are active on shorter substrates with one amino acid that is specific for the S1 binding subsite. That is, the insect chymotrypsins appear to have an extended substrate binding site requiring at least four amino acids for efficient catalysis. Recent studies have shown that chymotrypsins do respond to PI ingestion and are worthy of more detailed investigation. When larvae from H. armigera were fed on diets consisting of either potato proteinase inhibitor II, soybean trypsin inhibitor, aprotin (trypsin inhibitor) or potato proteinase inhibitor I, levels of chymotrypsin mRNA increased in all cases while trypsin mRNA decreased (Gatehouse et aL, 1997, supra). Other reports also mention upregulation of chymotrypsins in preference to trypsins (Bown et al., 1997, supra; Wu et al., Molecular Breeding 3: 371-380, 1997). Mazumdar-Leighton and Broadway (2001a, supra) assayed chymotrypsin activity in the gut of H. zea larvae and found that SBTI inhibited 95% of the chymotrypsin from the gut of control insects but only 35% of activity from the gut of insects that had prior exposure to SBTI in the diet.
Hence, consumption of proteinase inhibitors can lead to a drastic change in the complement of gut proteases which allows insects to adapt to the diet and survive. Changes in the complement of proteases after exposure to PIs have been detected in insects fed on both artificial diets and transgenic plants. The triggers that regulate these changes are still unknown and the responses vary with the species, the PI and its concentration, and the base diet. It is unclear why some inhibitors induce this response and others do not. It is clear, however, that some larvae are genetically pre-adapted to PIs, since prior exposure to a specific inhibitor is not necessary for an insect to be resistant (Broadway, 1996, supra).
There is a need to identify and investigate novel insect proteinases which are insensitive to PIs and to use these to screen for antagonists of the proteinases in order to develop agents useful in controlling insect growth, maintenance, development and/or survival.